The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Summary

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Abstract

Explant culture allows manipulation of developing organs at specific time points and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.

In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel's cartilage, nasal glands, tongue, and ear.

Introduction

For a number of experiments it is important to be able to culture tissue ex vivo to follow development. Culture of developing tissue provides access at defined periods of development and allows manipulation of genes by the addition of factors to the culture medium, or on loaded beads, and by the use of transfection and electroporation¹. For many experiments it is important to be able to visualize the tissue as it grows, for example, to follow the fate of lineage labeled cells as the tissue undergoes morphogenesis. This can be particularly problematic for tissues that develop deep within the embryo, which are not obvious when a block of tissue from ....

Protocol

1. Set Up

1. Sharpen the dissection instruments (using a sharpening stone lubricated with mineral oil) and sterilize prior to use for organ culture using a 70% ethanol spray and dry-heat sterilization. Sharpen the dissection needles using electrolysis in 2 M sodium hydroxide using a 12 V supply.
2. Prepare the culture medium used for the dissection and culture of embryonic organs, consisting of Advanced Dulbecco's Modified Eagle Medium F12 (DMEM F12) supplemented with 1% GlutaMAX and 1% penicillin-streptomycin.

2. Embryo Dissection

1. Cull a timed mated pregnant female using a schedule one method as ....

Results

In order to follow the movement of the first molar dental follicle, 250 μm frontal slices were taken through a mandible using the method described above. The dental follicle is the layer of mesenchyme that surrounds the outer enamel epithelium (OEE) of the developing tooth, and has previously been shown to take part in the formation of tissue of the periodontium ⁶. Mandibles were dissected at E14.5, the cap stage of tooth development. In the slice the outline of the dental epithelium was clear, and the condens.......

Discussion

This method of tooth culture has the advantage that access to the tooth germ is excellent, allowing accurate lineage tracing and placement of beads within the epithelium or the mesenchyme. Defined regions of the developing tooth germ can therefore be specifically targeted. During culture the changing morphology of the tooth germ can be followed, and the effect of manipulations quickly assessed.

The method, however, is only suitable for young tooth germs before substantial formation of hard tis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol	VWR	101077Y	100% ethanol was diluted in distilled H2O to 70%.
DMEM F12	Gibco	12634-010	Advanced Dulbecco's Modified Eagle Medium F12
GlutaMAX	Gibco	35050-061
Penicillin-streptomycin	Sigma	P0781
DiI (Molecular probes)	Vybrant	V-22885	Cell-labeling solution
	Invitrogen		Cell tracker CM-DiI, C-7000
DiO (Molecular probes)	Vybrant	V22886
Geminator 500	Thomas	Thomas No. 3885A20	Dry-heat sterilization
McIlwain tissue chopper	Ted Pella, Inc.	10180	Standard table
Organ culture dish (Center-Well Organ Culture Dish)	Falcon	353037
Membranes (Cell Culture Inserts, 0.4 μm pore size)	BD Falcon	353090	PET track-etched membrane, 6-well format
Metal grids (Stainless Steel - AISI 304 - Mesh)	Goodfellow	FE228710	(Fe/Cr18/Ni10)
AutoFlow Direct Heat CO2 Incubator	Nuaire	NU-5500
Picospritzer III	Intracel Ltd	051-0500-900 0-100 psi	Single channel picospritzer III
Glass capillary with filament 1 mm	WPI	TW100F-4
Tungsten wire 0.1 mm	Goodfellow	W005138
Tungsten wire 0.38 mm	Goodfellow	W005155
Aspirator tubes	Sigma	A5177	Used for mouth aspiration lineage tracers